Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for studying the spatial and temporal regulation of Rho GTPases. Early versions demonstrated that active RhoA was not restricted to the retracting rear and was activated at the leading edge of the migration front. In most of these FRET sensors, a Rho GTPase is directly tethered with an effector fragment and a FRET pair. Upon activation, the sensor adopts a closed/bound conformation and alters FRET. From a simple reaction-diffusion consideration, however, there is an intrinsic problem with this type of sensor. In the closed/bound conformation it temporarily loses the ability to interact with regulators or effectors and during this time can diffuse away from the initial site of activation. This type of sensor thereby loses fidelity in tracking signals, especially on small spatial scales. This issue was clearly demonstrated in our preliminary studies of Rac and Rho activation in adhesions and dendritic spines. Here we propose to address this issue by improving the dynamic range and off kinetics of the existing FRET sensors. In addition, a completely novel strategy - single molecule probes - will be developed to precisely capture active GEF, GAP and Rho-effector complexes in super resolution. Lastly, a potentially generalizable optogenetic strategy will be explored on signaling targets associated with Rho GTPases. These complementary imaging tools will provide a unique strength to resolve the spatiotemporal dynamics of signaling within minute subcellular structures.